Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein 1 (HYOU1) Suppressing Pathogenic Fibroblast Activation

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders. A multidisciplinary pipeline consisting of medicinal chemistry, molecular phenotyping, chemoproteomics, RNA-sequencing, short hairpin RNAs (shRNAs)-mediated gene silencing and in vivo studies led to a novel series of compounds suppressing pathogenic fibroblast activation via Hypoxia up-regulated protein 1 (HYOU1) inhibition. The first reported HYOU1 inhibitors are presented as a promising therapeutic approach in fibroblast-related diseases.+ image